Biomarkers associated with autoimmune diseases of the lung

ABSTRACT

The present disclosure is generally related to pulmonary autoantigens. The disclosure provides methods and kits for assessing whether a subject has or is predisposed to interstitial lung disease. Additionally the present disclosure provides methods of treatment and animal models of interstitial lung disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of PCT/US2011/040060, filed Jun. 10, 2011, which claims the benefit under 35 USC 119(e) of U.S. Provisional Patent Application No. 61/354,184, filed Jun. 11, 2010, each of which is incorporated herein by reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos. AI035297, DK59958, EY016408, and T32 HL007185 awarded by the National Institutes of Health. The government has certain rights in the invention.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 643662001200SeqList.txt, date recorded: Dec. 7, 2012, size: 14 KB).

FIELD

The present disclosure is generally related to pulmonary autoantigens. The disclosure provides methods and kits for assessing whether a subject has or is predisposed to interstitial lung disease. Additionally the present disclosure provides methods of treatment and animal models of interstitial lung disease.

BACKGROUND

Current methods for the treatment of autoimmune diseases of the lung rely on powerful immunosuppressive medications with significant systemic side effects. But validated lung-specific autoantigens, which may facilitate the diagnosis and treatment of autoimmune-mediated lung diseases, are currently unknown.

Therefore, a significant biomedical need exists for the identification of lung autoantigens and methods of their use as prognostic or diagnostics biomarkers for autoimmune diseases of the lung. A need also exists for antigen-specific therapies for autoimmune disease of the lung, such as interstitial lung disease.

SUMMARY

The present disclosure is generally related to pulmonary autoantigens. The disclosure provides methods and kits for assessing whether a subject has or is predisposed to interstitial lung disease. Additionally the present disclosure provides methods of treatment and animal models of interstitial lung disease.

In particular, the disclosure provides diagnostic and prognostic tests for interstitial lung diseases (ILD) by quantifying antibody or T-cell mediated immune responses to a pulmonary autoantigen (e.g., antigen expressed in the lung that has at least one bactericidal/permeability-increasing protein (BPI) domain. In some embodiments, the methods involve assessing whether a mammalian patient has or is predisposed to an interstitial lung diseases (ILD), comprising: a) subjecting a biological sample from the patient to a procedure for quantitation of an immune response to a lung autoantigen, wherein the lung autoantigen comprises at least one bactericidal/permeability-increasing protein (BPI) domain, and wherein the procedure comprises an antibody-based assay or a T cell-based assay; and b) detecting an elevated immune response to the lung autoantigen in the biological sample as compared to a control biological sample, wherein the elevated immune response is associated with presence of the ILD or a predisposition to ILD. Preferred examples of such lung autoantigens include long palate, lung, and nasal epithelium carcinoma-associated protein 1 (LPLUNC1), LPLUNC1-like proteins, vomeromodulin, and vomeromodulin-like proteins. Lung autoantigens of this disclosure may be measured in a biological sample including but not limited to blood, plasma, serum, bronchial alveolar lavage (BAL) fluid, and lung tissue. In some embodiments, the ILD is idiopathic ILD or connective tissue disease associated ILD. Patients subjected to the methods may have or may be suspected of having a systemic autoimmune disease, such as rheumatoid arthritis, scleroderma, sjogren's syndrome, systemic lupus erythematosis, sarcoidosis, wegener's granulomatosis, or autoimmune polyendocrine syndrome type 1 (APS-1).

One aspect of the disclosure provides an antibody-based assay for measurement of lung autoantigen-reactive antibodies present in the biological samples of the patient for use in the methods of a preceding paragraph. Exemplary antibody-based assays include ELISA, Western blotting, immunofluoresence analysis, flow cytometry, and antibody microarray. Additionally kits for use in the antibody-based methods are provided. In some embodiments, the kits comprise a first reagent that specifically binds to the lung autoantigen-reactive antibodies, and a second reagent for detecting the antibodies, wherein the first reagent is the lung autoantigen or a cell expressing the lung autoantigen, and the second reagent comprises a secondary antibody that is reactive with constant regions of the lung autoantigen-reactive antibodies of the mammalian subject.

Another aspect of the disclosure provides a T cell-based assay for measurement of lung autoantigen-reactive T lymphocytes present in the biological sample of the patient for use in the methods of a preceding paragraph. Exemplary T cell-based assays include cytokine ELISA, ELISPOT analysis, flow cytometry, and proliferation assay. Additionally, kits for use in the T cell-based methods are provided. In some embodiments, the kits comprise a first reagent that specifically activates the lung autoantigen-reactive T lymphocytes, and a second reagent for detecting the activated T lymphocytes, wherein the first reagent comprises the lung autoantigen or a peptide derived therefrom, and the second reagent comprises thymidine.

Moreover the present disclosure provides methods that further comprise one or more additional steps. In some embodiments, the methods of a preceding paragraph further comprise performing one or both of a pulmonary function test and a high resolution computed tomography scan on the patient. In some embodiments, the methods of a preceding paragraph further comprising: c) administering a treatment to the patient when the elevated immune response is detected. In one aspect, the treatment comprises one of the group consisting of a corticosteroid, cyclophosphamide, mycophenolate mofetil, and azathioprine. In another aspect the treatment comprises an antigen-specific tolerance regimen. In some embodiments, the treatment comprises a mucosal tolerance regimen comprising dispensing a formulation to the patient by an oral or an intra-nasal route, wherein the formulation comprises a pharmaceutically acceptable excipient, and an effective amount of the lung autoantigen, or a peptide derived therefrom. In some embodiments, the treatment comprises a parenteral tolerance regimen comprising dispensing a formulation to the patient by intravenous or subcutaneous injection, wherein the formulation comprises an effective amount of the lung autoantigen, a peptide derived therefrom, or a nucleic acid encoding the lung autoantigen in operable combination with a regulatory sequence. In some embodiments, the treatment comprises an antigen-coupled cell tolerance regimen comprising dispensing a formulation to the patient by intravenous injection, wherein the formulation comprises an effective amount of the lung autoantigen, or a peptide derived therefrom, wherein the autoantigen or the peptide is coupled to ethylene carbodiimide-fixed, autologous antigen presenting cells. In some embodiments, the treatment comprises a regulatory T cell regimen comprising dispensing a formulation to the patient by intravenous injection, wherein the formulation comprises an effective amount of ex vivo-expanded lung autoantigen-specific regulatory T cells.

The disclosure further provides an animal model of interstitial lung disease comprising a mammal immunized with a formulation comprising an adjuvant and a lung autoantigen comprising at least one bactericidal/permeability-increasing protein (BPI) domain. In one aspect, the lung autoantigen is a long palate, lung, and nasal epithelium carcinoma-associated protein 1 (LPLUNC1) or a LPLUNC1-like protein. In one aspect, the autoantigen is vomeromodulin or a vomeromodulin-like protein. In one aspect the mammal is selected from the group consisting of rodents, dogs, cows, and non-human primates. In one aspect the mammal is a mouse or a rat.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a representative plot of CD4⁺ lung lymphocytes from a BALB/c Aire^(o/o) mouse at 14 weeks, showing IL-17A, IFN-γ, IL-4 and IL-10 containing cells. The right panel shows percentages of total CD4⁺ lung lymphocytes producing cytokines averaged from 5 BALB/c Aire^(o/o) mice aged 12-16 weeks. Data are mean±SEM. FIG. 1B depicts an indirect immunofluorescence stain with serum from a NOD Aire^(o/o) mouse with pulmonary disease on frozen lung section from a immunodeficient SCID mouse. The lower panel depicts a higher magnification image of the lung section shown in upper right panel: Green, serum staining; blue, staining with nuclear marker 4′,6′-diamidino-2-phenylindole (DAPI).

FIG. 2 characterizes lung lymphocytes in Aire^(0/0) mice. The representative FACS plot is gated on lung lymphocytes from a NOD Aire^(0/0) mouse at 10 weeks. Similar percentages were seen in both younger and older mice, as well as in BALB/c Aire^(0/0) mice.

FIG. 3 demonstrates that vomeromodulin (VM) is the predominant antigen targeted in lungs of Aire^(o/o) mice. FIG. 3A shows an immunoblot of whole lung lysate probed with sera from BALB/c Aire^(o/o) mice aged 8-20 weeks, the individual animals are numbered. The blot revealed an 80 kD antigen target. FIG. 3B depicts an immunoblot of BAL fluid probed with sera from BALB/c Aire^(o/o) mice, which also revealed the 80 kD antigen. FIG. 3C depicts an immunoblot of BAL fluid probed with NOD mice bled serially and BALB/c mice sacrificed at various ages. In FIG. 3D sera from Aire^(o/o) mice were used to immunoprecipitate the antigen from BAL fluid, which was then run on a Coomassie-stained 2D gel. Three spots at 80 kD migrated near an isoelectric point ˜5.5, as indicated by the arrow. All spots were analyzed by mass spectrometry. FIG. 3E depicts the amino acid sequence of the 80 kD spot, indicating that it is vomeromodulin (SEQ ID NO:1). The cDNA of vomeromodulin is set forth as GENBANK Accession No. NM_(—)001025574.1. Identified peptides, were mapped onto the VM amino acid sequence and revealed coverage of nearly the entire protein. FIG. 3F shows a competition blot, which was to confirm autoantibody reactivity to VM. The blot showed that 80 kD reactivity was abolished after addition of recombinant VM-MBP. The MBP tag alone failed to abolish reactivity. FIG. 3G shows the staining of the apical surface of the bronchiolar epithelium and to rare cells within lung alveoli with both Aire^(o/o) serum and a rat VM anti-serum.

FIG. 4 shows an immunoblot of vomeromodulin after treatment with N-glycosidase. The immunoblot of BAL fluid was probed with NOD Aire^(0/0) mouse serum before and after treatment with N-glycosidase. The amount of post-treatment BAL analyzed was ˜155 of the pre-treatment protein.

FIG. 5 shows T cells with specificity for vomeromodulin in Aire^(o/o) mice. FIG. 5A shows a RT-PCR of vomeromodulin cDNA after 35 cycles in indicated tissues. The RT-PCR revealed a band at expected size of 1.8 kb in lung only. The DNA band was excised and sequenced, confirming that full length VM cDNA was amplified. FIG. 5B shows an immunoblot using BALB/c Aire^(o/o) serum to probe tissue lysates of indicated organs and recombinant VM-MBP. Reactivity to the 80 kD band was only seen in lung lysate. Reactivity to VM-MBP occurred at the expected weight of 100 kD. FIG. 5C shows representative results from two independent experiments in which TEC stroma from Aire^(o/o) and Aire^(+/+) thymi was assayed in quadruplicate for VM, insulin 2 (Ins2) and glutamic acid decarboxylase 67 (GAD67) by real-time PCR; data are normalized expression relative to wild-type±SD. FIG. 5D shows an ELISPOT analysis of IFN-γ producing T cells in Aire^(o/o) and Aire^(+/+) BALB/c mice aged 10-14 weeks (*P=0.005). The Y axis indicates number of spots per 10,000 T cells.

FIG. 6 illustrates the induction of lung-specific disease by breaking tolerance to vomeromodulin in wild-type mice. FIG. 6A shows H&E stains of lungs from BALB/c WT mice immunized with VM-MBP or MBP. FIG. 6B shows that four of six mice immunized with VM-MBP exhibited lung disease, scored as shown. Mononuclear cell infiltrates were limited to the lung, except in one mouse immunized with MBP with salivary infiltrates. Lines indicate mean disease scores. FIG. 6C represents results of a VM autoantibody assay, showing a VM-specific immune response in mice immunized with VM-MBP, but not in the MBP immunized controls.

FIG. 7 shows the results of an ELISPOT analysis of T cells in Aire^(0/0) mice. The ELISPOT analysis of IFN-γ-producing T cells in two BALB/c Aire^(0/0) mice 12 weeks of age immunized with full length VM is presented. The Y axis indicates number of spots per 90,000 CD4⁺ T cells.

FIG. 8 illustrates the lung-specific disease phenotype observed after adoptive transfer of VM-specific T cells. FIG. 8A introduces the protocol for adoptive transfer beginning with immunization of BALB/c WT mice with a VM or Ova peptide. Ten days later, lymph node and spleen cells were activated in vitro with respective peptides. Activated cells were analyzed in a proliferation assay or transferred into BALB/c SCID mice. FIG. 8B shows results of representative [H³] thymidine incorporation assays in cells harvested from immunized mice. Each condition was performed in triplicate; data are mean±SEM. The differences between Ova or VM peptides and scramble controls are statistically significant (*P<0.05 for all comparisons, two tailed t-test). FIG. 8C displays H&E stains and histology analyses for organs harvested 4-6 weeks after VM specific lymphocytes were transferred into mice. The lung images revealed a mononuclear peribronchovascular infiltrate in the mouse receiving VM specific cells. FIG. 8D displays disease scores of mice after adoptive transfer (AT) of antigen specific cells.

FIG. 9 documents autoreactivity to a human bronchial epithelial protein, LPLUNC1, in a patient with APS1 and lung disease. FIG. 9A shows an immunofluorescence stain of normal frozen human lung with serum from an APS1 patient with lung disease, whereas FIG. 9B shows results for a normal healthy patient. Green coloring represents serum staining, blue coloring represents staining with DAPI. FIG. 9C reviews the genomic organization of the human VM pseudogene locus (C20orf115) and shows the adjacent human PLUNC gene family with individual genes numbered as indicated. FIG. 9D illustrates the domain structure of the murine vomeromodulin protein showing the BPI domain. FIG. 9E illustrates the domain structure of human LPLUNC1, also including a BPI domain. FIG. 9F shows results of an autoantibody assay, detecting autoantibodies to LPLUNC1 in serum from an APS1 patient with lung disease (n=1), healthy controls (n=11) and APS1 patients without lung disease (n=11). The assay was run in triplicate using in vitro transcribed and translated, radiolabeled human LPLUNC1 protein. As a positive control, two commercial anti-human LPLUNC1 antibodies were run. Representative results from 2 independent experiments are shown. FIG. 9G shows normal frozen human lung stained by immunofluorescence with antibody to human LPLUNC1. FIG. 9H depicts a high magnification view of a normal frozen human lung after immunofluorescence staining and shows the co-localization (bottom left) of serum from the APS1 patient with lung disease (top left, red) and the LPLUNC1 antibody (top right, green) on the bronchiolar epithelium. A serial lung section stained with healthy patient serum did not demonstrate autoreactivity.

FIG. 10 shows the results of a KCNRG autoantibody assay in the APS1 patient with lung disease.

FIG. 11 provides a plot of the LPLUNC1 autoantibody index of three groups of subjects. Autoantibodies to human LPLUNC1 in serum from patients with ILD (n=186), normal control patients (n=54) and positive controls (n=3) were detected in an autoantibody assay run in triplicate using in vitro transcribed and translated, radiolabeled human LPLUNC1 protein.

FIG. 12 provides the results of an autoantibody assay, detecting autoantibody to LPLUNC1 in serum from human subjects. The values shown in white boxes correspond to APS-1 patients with known interstitial lung disease. For comparison, patients with other known inflammatory lung disorders were also screened. None of the asthma or chronic obstructive pulmonary disease patients demonstrated autoreactivity to LPLUNC1. The assay was run using in vitro transcribed and translated, radiolabeled human LPLUNC1 protein.

FIG. 13 provides results of an autoantibody assay, detecting autoantibody to LPLUNC1 in serum from human subjects with interstitial lung disease (n=153) and in normal controls (n=112). The autoantibody assay was run in duplicate using in vitro transcribed and translated, radiolabeled human LPLUNC1 protein. The interstitial lung disease (ILD) sera are from patients seen and enrolled in the clinical database of the UCSF ILD clinic. The normal controls were selected to age and race match the ILD patients as closely as possible, and are subjects who do not have known interstitial lung disease. The horizontal line depicts a value above which the specificity of the assay equals 100%, as determined by calculating the Receiver Operator Curve.

DEFINITIONS

To facilitate an understanding of the embodiments disclosed herein, a number of terms and phrases are defined below.

The terms “interstitial lung disease” and “ILD” as used herein refer to a group of lung diseases affecting the interstitium (the tissue and space around air sacs of the lungs). ILDs affect areas in the lung comprising the alveolar epithelium, pulmonary capillary endothelium, basement membrane, perivascular and perilymphatic tissues. The term ILD is used to distinguish these diseases from obstructive airways diseases.

The term “predisposed to” as used herein in connection with a disease, refers to an increased statistical likelihood that test subjects having a specific phenotype, such as an elevated immune response to an antigen, will develop the disease as compared to control subjects that do not possess the specific phenotype.

The terms “bacterial/permeability-increasing protein,” “BPI,” “lipopolysaccharide-binding protein,” “LBP,” cholesteryl ester transfer protein,” and “CETP” as used herein refer to a human antigen, as well as its cDNA and genomic DNA, and mammalian counterparts thereof. The cDNA sequence of human BPI is provided by GENBANK Accession No. AX752831.1. The amino acid sequence of human BPI is provided below as SEQ ID NO:3

MRENMARGPCNAPRWVSLMVLVAIGTAVTAAVNPGVVVRISQKGLDYASQQGTAAL QKELKRIKIPDYSDSFKIKHLGKGHYSFYSMDIREFQLPSSQISMVPNVGLKFSISNANIKI SGKWKAQKRFLKMSGNFDLSIEGMSISADLKLGSNPTSGKPTITCSSCSSHINSVHVHISK SKVGWLIQLFHKKIESALRNKMNSQVCEKVTNSVSSKLQPYFQTLPVMTKIDSVAGINY GLVAPPATTAETLDVQMKGEFYSENHHNPPPFAPPVMEFPAAHDRMVYLGLSDYFFNT AGLVYQEAGVLKMTLRDDMIPKESKFRLTTKFFGTFLPEVAKKFPNMKIQIHVSASTPP HLSVQPTGLTFYPAVDVQAFAVLPNSSLASLFLIGMHTTGSMEVSAESNRLVGELKLDR LLLELKHSNIGPFPVELLQDIMNYIVPILVLPRVNEKLQKGFPLPTPARVQLYNVVLQPH QNFLLFGADVVYK.

As used herein, the term “BPI domain” refers to a structural unit within a larger polypeptide, which shares its three dimensional architecture and at least 40% amino acid sequence identity with a family of secretary proteins having antimicrobial activity. The human BPI protein has two BPI domains: BPI1 from residues 35 to 260; and BPI2 from residues 284 to 484. The conserved domain search (CD-Search) of the National Center for Biotechnology Information (NCBI) using default parameters can be used to determine whether the amino acid sequence of a protein of interest possesses a BPI domain (Marchler-Bauer and Bryant, Nucleic Acids Res, 32(W)327-331, 2004; and Marchler-Bauer et al., Nucleic Acids Res, 37(D)205-10, 2009).

The terms “long palate, lung and nasal epithelium carcinoma-associated protein 1,” “LPLUNC1,” “von Ebner minor salivary gland protein,” “VEMSGP” and “c20orf114” as used herein refer to a human pulmonary antigen, as well as its cDNA and genomic DNA, and mammalian counterparts thereof. LPLUNC1 is thought to play a role in the innate immune response to bacterial exposure in the mouth, nasal cavities and lungs. The cDNA sequence of human LPLUNC1 is provided by GENBANK Accession No. NM_(—)033197.2. The amino acid sequence of human LPLUNC1 is provided below as SEQ ID NO:2: MAGPWTFTLLCGLLAATLIQATLSPTAVLILGPKVIKEKLTQELKDHNATSILQQLPLLS AMREKPAGGIPVLGSLVNTVLKHIIWLKVITANILQLQVKPSANDQELLVKIPLDMVAG FNTPLVKTIVEFHMTTEAQATIRMDTSASGPTRLVLSDCATSHGSLRIQLLHKLSFLVNA LAKQVMNLLVPSLPNLVKNQLCPVIEASFNGMYADLLQLVKVPISLSIDRLEFDLLYPAI KGDTIQLYLGAKLLDSQGKVTKWFNNSAASLTMPTLDNIPFSLIVSQDVVKAAVAAVL SPEEFMVLLDSVLPESAHRLKSSIGLINEKAADKLGSTQIVKILTQDTPEFFIDQGHAKVA QLIVLEVFPSSEALRPLFTLGIEASSEAQFYTKGDQLILNLNNISSDRIQLMNSGIGWFQPD VLKNIITEIIHSILLPNQNGKLRSGVPVSLVKALGFEAAESSLTKDALVLTPASLWKPSSP VSQ. As used herein, the term “LPLUNC1-like protein” refers to members of the PLUNC superfamily. In preferred embodiments, LPLUNC-1 like proteins are at least 70% identical to the amino acid sequence of SEQ ID NO:2.

The terms “vomeromodulin” and “VM” as used herein refer to mouse pulmonary antigen, as well as its cDNA and genomic DNA, and mammalian counterparts thereof. The cDNA sequence of mouse vomeromodulin is provided by GENBANK Accession No. NM_(—)001025574.1. The amino acid sequence of mouse vomeromodulin is provided below as SEQ ID NO:1: MWVLQALAIMLSIQAGTLDLVETPPVVGNLPVAMPVPLNLPVGGLSPPVLKGPVNHQM LPPKRPVPPPKGGKCAPAARYFLSSDKLHDYLMSTLPPQIEDMVKCDEVNLEGMLADV LNTVESSDLLSLLDGISLLKGGEGGGLGIGGLLGNEGNGDSSKPSSGSKATGGLGQLIPG GIPGTEALGGLLNLGGDKSSGKGLLNGDGLSKIKKPLEDAVENVSGIKDAIQEKVNEVV PDGVKEPLNDVLKMDIKDTLLELKVGQVTLDDMEINMEANGMQVLSMLTATIDGKGV LGPVISLLQFEAKMDVMTTIAVASNNTQCVNLDAQDTHMHVKEMKIQLVETVTGKVP LPVPLPLDQIIPAIVTAKINENLEKSNSCAIVLNDFNNCKNNTGLFSYQVNTARISPKGLVI LYCAKANIGNKTVPVPGGRLPPDPKNASIAVTISSTTLKTLVKEVAKNSSVQMDGLEAQ ITHIAFASQENNTLRVVYKVDITKNGEHFATGETKLFISHGSKISNSTLIPDVKLIRSEHSV VPPEAKEEVEGILSEVGKVAWSNFNETYKKMNIPVGVSSHTLKNSDVKLMKSIDLQAA S. As used herein, the term “vomeromodulin-like protein” refers to proteins that are at least 70% identical to the amino acid sequence of SEQ ID NO:1.

The terms “autoimmune polyendocrine syndrome type 1” and “APS-1” as used herein refers to an autosomal recessive autoimmune disease phenotype caused by a defect in the AutoImmune Regulator (AIRE) transcription factor.

The term “pulmonary function test” as used herein refers to any diagnostic test, such as spirometry, or medical examination designed to assess the functional integrity of the lung. Typically, such tests comprise determinations of the volume of air that can be inhaled and exhaled and the rates of the respective airflow.

The term “high resolution computer tomography” as used herein refers to a medical imaging technology for the non-invasive diagnosis of living human or animal subjects, wherein the technology provides a three-dimensional representation of the organs in question and allows for the determination of the organs' anatomical and functional integrity.

The term “animal model” as used herein refers to a non-human animal that mimics a human disease phenotype in the sense that treatment measures which remedy the disease phenotype in the non-human animal can also remedy the disease phenotype in human patients.

The term “adjuvant” as used herein refers to a composition that acts to facilitate a specific antibody based and/or cell based immune response against an antigen without itself being a specific target of the immune response.

The term “elevated level” as used herein in connection to an immune response, refers to an antigen-specific antibody and/or T cell response (e.g., vomeromodulin or LPLUNC1 reactive immune responses) in a biological sample that is at least 2.5-fold greater than the level of the immune response to the antigen in a control sample. In some embodiments, an elevated level is at least 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10-fold greater.

As used herein the term “antibody-based assay” refers to any technique that involves the use of an antibody to detect an antigen. Such techniques include but are not limited to immunostaining, ELISA, antibody microarray, flow cytometry, and Western blotting.

As used herein the term “cell-based assay” refers to any technique that involves the measurement of antigen-specific T lymphocytes. Such techniques include but are not limited to ELISPOT assays, proliferation assays, and MHC-peptide tetramer staining.

As used herein, the term “microscopy-based assay” refers to any technique that involves the use of a microscope to assess a phenotype of a cell or tissue.

DETAILED DESCRIPTION

The present disclosure is generally related to pulmonary autoantigens. The disclosure provides methods and kits for assessing whether a subject has or is predisposed to interstitial lung disease. Additionally the present disclosure provides methods of treatment and animal models of interstitial lung disease.

The lung is often damaged in systemic autoimmune diseases such as rheumatoid arthritis and scleroderma. One of the most common pulmonary manifestations of systemic autoimmune syndromes is interstitial lung disease (ILD). This term comprises a heterogeneous group of disorders in which fibrosis and inflammation occur within alveolar walls or in the loose tissue surrounding peribronchovascular sheaths, interlobular septa and the visceral pleura. An alternative form of interstitial lung disease is idiopathic ILD, which occurs in the absence of an autoimmune syndrome and instead arises as an isolated pulmonary process.

The AIRE-deficient model of autoimmunity. Human subjects with defects in the autoimmune regulator (AIRE) develop Autoimmune Polyglandular Syndrome Type 1 (APS1), a multi-organ autoimmune disease that involves the lung in some subjects. AIRE is expressed primarily within the thymus in thymic medullary epithelial cells (mTECs). AIRE promotes immune tolerance by driving the ectopic expression of a wide array of organ specific self-antigens in mTECs. In the absence of appropriate AIRE expression, these self-antigens are not displayed in the thymus, leading to a defect in thymic deletion of autoreactive T cells. The multi-organ nature of disease in AIRE-deficient animals is viewed as a result of the spectrum of self-antigens whose thymic expression relies on AIRE. Aire-deficient mice (Aire^(o/o)) develop lung autoimmunity that is strikingly similar in pattern to the disease reported in APS1 patients, but the specificity of this response was unknown.

Several important findings were made relating to the lung-specific autoimmune reactivity observed in Aire^(o/o) mice and human APS1 patients. First, vomeromodulin was identified as the predominant lung-specific autoantigen in Aire^(o/o) mice Immunoblots using lung lysate prepared from immunodeficient SCID mice and probed with serum from Aire^(o/o) mice demonstrated the presence of autoantibodies in Aire^(o/o) mice that specifically targeted vomeromodulin (VM), a 80 kD lung protein. VM was located on the surface of the bronchiolar epithelium. It was shown to be secreted into the airway and detectable in BAL fluid from the lung by immunoblotting. No other lung autoantigens besides VM were identified, demonstrating that VM is the primary lung antigen in Aire^(o/o) mice. Importantly, recombinant VM protein was shown to neutralize autoreactive antibodies found in Aire^(o/o) mice, suggesting a therapeutic utility for purified VM. Additionally, Aire^(o/o) mice were shown to have autoreactive T cells that specifically respond to VM and that either the adoptive transfer of VM reactive T cells into SCID mice or the immunization of wild type mice with purified VM can cause lung-specific autoimmune disease phenotypes. These experiments therefore demonstrated that the lung-specific autoantigen VM alone can act as a disease causing agent. The neutralization of cellular and antibody based autoimmune reactivity against VM can therefore be of therapeutic use in the treatment of lung-specific autoimmune diseases and aid in the development of Aire^(o/o) mice as an animal model for pharmacological research.

The human homolog of mouse VM was determined to be a pseudogene, which is not expressed in human APS1 patients. Nevertheless, the organization of the human genomic region surrounding this pseudogene is similar to the genomic organization around mouse VM and comprises coding sequences for PLUNC proteins. Specifically, LPLUNC1 is encoded in a region immediately adjacent to the human VM pseudogene. Like VM, LPLUNC1 is highly expressed in the bronchial epithelium of the human lung and serum of a human APS1 patient was successfully used to stain LPLUNC1 in lung tissue obtained from a normal control specimen. The pattern of immunoreactivity was nearly identical to the pattern of staining seen using sera from Aire^(o/o) mice. This data shows that the lung disease in an APS1 patient closely recapitulates the lung disease in Aire^(o/o) mice, suggesting that LPLUNC1 is an important human lung autoantigen.

Prognostic and Diagnostic Methods

In one embodiment, patients at risk of or suspected of having ILD and having an elevated immune response to a lung autoantigen (pulmonary antigen such as PLUNC1, which comprises at least one BPI domain) are identified as having or being predisposed to ILD. In some embodiments, the patient has a systemic autoimmune disease. In some embodiments, the methods are performed on more than one occasion in order to assess a patient's response to therapy or to assess a patient's prognosis.

In one embodiment, background immune response to the lung autoantigen in normal subjects are established to define an ‘elevated’ immune response level of expression of the marker. Depending on the technique used and the immune response examined, different values may be used to define an ‘elevated’ immune response to the lung autoantigen. In order to define an ‘elevated’ immune response, statistical analysis may be used.

Antibody Based Methods

In some embodiments, an elevated antibody response to a lung autoantigen (pulmonary antigen such as PLUNC1, which comprises at least one BPI domain) in a biological sample from a patient is detected. Antibody-based methods include various techniques that involve the measurement of lung autoantigen (pulmonary antigen such as PLUNC1, which comprises at least one BPI domain)-reactive antibodies. Commonly used antibody-based techniques to detect the level of a humoral immune response of a patient include but are not limited to ELISA, Western blotting, immunofluorescence analysis, flow cytometry, and antibody microarray.

T Cell-Based Methods

In some embodiments, an elevated T cell responses to a lung autoantigen (pulmonary antigen such as PLUNC1, which comprises at least one BPI domain) in a biological sample from a patient is detected. T Cell-based methods include various techniques that involve the measurement of T cell activation by the lung autoantigen. Commonly used T cell-based techniques to detect the level of a cellular immune response of a patient include but are not limited to proliferation assays, and cytokine secretion assays (e.g., ELISA, ELISPOT, etc.). Cytokines that are frequently used to assay T cell activation include but are not limited to IL-2, and interferon-gamma.

Treatment Methods

In yet another embodiment, when an elevated immune response to a lung autoantigen (e.g., pulmonary antigen such as PLUNC1, which comprises at least one BPI domain) is detected in a biological sample from a patient as compared to that measured in a control biological sample, treatment of the patient is indicated. For instance, in some embodiments, upon detection of an elevated immune response to a lung autoantigen (pulmonary antigen such as PLUNC1, which comprises at least one BPI domain) a non-specific immunosuppressive treatment or an lung autoantigen-specific tolerance regimen is administered. Suitable non-specific immunosuppressive treatments include but are not limited to corticosteroid, cyclophosphamide, mycophenolate mofetil, azathioprine, and mTOR inhibitor (e.g., rapamycin). In some preferred embodiments, the lung autoantigen is in the form of a soluble protein, a protein fixed to an antigen presenting cell, a soluble peptide, a peptide fixed to an antigen presenting cell or a peptide presented by a soluble MHC multimer. Suitable antigen-specific tolerance regimens include but are not limited to mucosal tolerance, parenteral tolerance, and antigen-coupled cell tolerance regimens (See, e.g., Miller et al., Nature Reviews Immunology, 7:665-677, 2007; and Ludvigsson et al., N Engl J Med, 359:19019-1920, 2008). Alternatively, a regulatory T cell regimen is employed, which involves the ex vivo expansion of autologous lung autoantigen-specific regulatory T cells.

Kits

In another embodiment, a kit comprising a lung autoantigen (pulmonary antigen such as PLUNC1, which comprises at least one BPI domain) is provided. In some embodiments, the kit further comprises a reagent for detecting antibody binding to the lung autoantigen. In other embodiments, the kit further comprises a reagent for detecting activation of T lymphocytes by the lung autoantigen. Reagents capable of detecting antibody binding are typically directly or indirectly linked to a tag such as a radiolabel, a chromophore, a fluorophore or an enzyme that catalyzes a reaction resulting in a detectable signal. Reagents capable of detecting activation of T lymphocytes by the lung autoantigen also typically comprise a tag.

EXAMPLES

The present disclosure is described in further detail in the following examples, which are not in any way intended to limit the scope of the disclosure as claimed. The attached figures are meant to be considered as integral parts of the specification and description of the disclosure. The following examples are offered to illustrate, but not to limit the claimed disclosure.

In the experimental disclosure which follows, the following abbreviations apply: M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); μg (micrograms); pg (picograms); L (liters); ml and mL (milliliters); μl and μL (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient); ND (not done); NA (not applicable); rpm (revolutions per minute); H₂O (water); dH₂O (deionized water); aa (amino acid); bp (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); OD (optical density); PCR (polymerase chain reaction); and RT-PCR (reverse transcription PCR). Additional abbreviations include: BAL (bronchoalveolar lavage) fluid; BPI (bacterial permeability-increasing protein); LPLUNC (long palate, lung and nasal epithelium carcinoma-associated protein); MHC (major histocompatibility complex); Th (helper T lymphocyte); and VM (vomeromodulin).

Example 1 Lung-Specific Biomarkers for the Diagnosis and Treatment of Autoimmune Disease

This example provides a description of the materials and methods and results of analyses of the role of vomeromodulin and LPLUNC1 as predominant antigens in autoimmune diseases of the lung.

Materials and Methods

Mice: Aire^(o/o) mice were generated as described (1, 2). Aire^(o/o) mice used in these experiments were backcrossed into the C57BL/6, BALB/c, and NOD ShiLt/J backgrounds>10 generations. BALB/c SCID mice were purchased from the Jackson Laboratory. All mice were housed in a pathogen-free barrier facility at UCSF. Experiments complied with the Animal Welfare Act and NIH guidelines for the ethical care and use of animals in biomedical research and were approved by the UCSF Animal Care and Use Committee.

Histology: Organs from mice were harvested and fixed overnight in 10% formalin, embedded in paraffin, sectioned, and stained for hematoxylin and eosin. Human specimens were stained by the UCSF Pathology Laboratory Immune infiltrates of organs were confirmed by an independent reading of the slides with a blinded observer.

Lung histology scoring system: Lung histology sections were scored based on the following grading system: grade 0, normal lung; grade 1, infrequent perivascular and peribronchiolar mononuclear infiltrates; grade 2, frequent perivascular and peribronchiolar mononuclear infiltrates; grade 3, dense perivascular and peribronchiolar mononuclear infiltrates and interstitial pneumonia; grade 4, diffuse perivascular and peribronchiolar mononuclear infiltrates, interstitial pneumonia and architectural distortion.

Immunostaining: Immune cell subtypes were visualized by immunohistochemistry using antibodies specific for CD4, CD8, and B220 (BD Biosciences) and a DAB staining kit (Vector Laboratories) on 7-micron frozen sections of Aire^(o/o) mice cut on a cryostat. The human lung tissue biopsy was stained by the UCSF Pathology Laboratory.

Indirect immunofluorescence: Seven-micron frozen sections from SCID mice and a lung tissue sample provided by the UCSF Pathology Department taken from a patient who expired of non-pulmonary disease were fixed and blocked in PBS containing 1% bovine serum albumin and 3% serum from same species as the secondary antibody, overnight at 4° C. Primary incubation with mouse sera was at 1:100 for an hour and goat anti-mouse FITC (Jackson Immunoresearch) was used at 1:1000 for an hour. Primary incubation using human sera was at 1:100 for an hour and secondary incubation with donkey anti-human FITC (Jackson Immunoresearch) was at 1:2000 for an hour. An anti-LPLUNC1 antibody (Abnova, No. H00092747-B01P) was used at 1:100 and incubated for an hour and goat anti-mouse FITC (Jackson Immunoresearch) was used at 1:1000 for an hour. Slides were examined on a microscope (Axiostar; Carl Zeiss MicroImaging, Inc.) with 5″, 10″, 20″ and 40″ lenses. Images were obtained using an AxioCam with AxioVision software (both from Carl Zeiss MicroImaging, Inc.).

Flow cytometry: Lungs were minced and then digested in 2 mg/ml collagenase Dulbecco's Modified Eagle Medium (DMEM) for 20 minutes. The remaining tissue was dispersed by vortexing and filtered through nylon mesh. Cells were placed in DMEM complete media with 10% FCS and Golgi-Stop (BD Biosciences) and stimulated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) and 0.5 μM ionomycin (Sigma-Aldrich) for 4 h at 37° C. After the incubation, cells were surface stained with antibodies specific for CD4, CD8, and CD45 to be able to sort lymphocytes, then permeabilized and stained with antibodies specific for IL-4, IL-10, IL-17A, IFN-γ, or isotype control (BD Biosciences). Cells were analyzed on a LSRII flow cytometer (BD Biosciences) (3).

Immunoblotting: Sera were screened for the presence of autoantibodies by immunoblotting as described (2). Sera from BALB/c, B6 and NOD Aire^(o/o) and Aire^(+/+) mice were used at a 1:600 dilution and incubated overnight. The secondary antibody was peroxidase-conjugated goat antibody to mouse IgG (Jackson Immunoresearch) used at 1:20,000 on immunoblots (1:15,000 on multiscreen immunoblots) for 1 hr. For competition studies, sera were pre-incubated with serial dilutions of recombinant VM-MBP or MBP overnight at 4° C. The anti-VM sera were used at 1:200 for 1 hr, and a peroxidase-conjugated goat antibody to rabbit IgG secondary antibody was used at 1:10,000 for 1 hr. For the multi-organ blot protein loading control, a GAPDH antibody (Santa Cruz Biotech) was used at 1:1000, and a peroxidase-conjugated goat antibody to mouse IgG secondary (Jackson Immunoresearch) antibody was used at 1:2000 for 1 hr. Tissue lysates were prepared in 0.1% CHAPS buffer from frozen organs harvested from immunodeficient SCID mice.

Immunoaffinity purification: Immunoprecipitation of the VM autoantigen was performed with Aire^(o/o) sera as described (2). The autoantigen was isolated from BAL fluid obtained by lavaging with PBS the lungs of several perfused and bled SCID mice. Protein agarose A/G beads (1:1 ratio) were covalently coupled to Aire^(o/o) serum that had known reactivity to the 80 kD protein and placed into a column. The columns were washed in 30 mL PBS, and BAL was passed through the matrix. Columns were washed with 30 mL PBS and washed again with 30 mL of 10 mM phosphate, pH 6.8. Column elutions were collected by passing 0.5 ml fractions of 100 mM glycine, pH 2.5, over the column and collecting the flow-through. Elutions from multiple runs were pooled and concentrated in a centrifugal protein concentrator (Sartorius). The isolated protein was run out on a pH 3-10, non-linear two-dimensional gel in MOPS buffer, coomassie stained and the excised spots were sent for peptide mass spectrometry analysis.

Preparation of cDNA from organs and RT-PCR: RNA from organs was prepared according to manufacturer's instructions with an RNA isolation kit (Stratagene). RNA was reverse transcribed into cDNA using Superscript III (Invitrogen) and poly-dT primers and the VM and cyclophilin cDNA was amplified for 35 cycles. The following VM primers were used: forward 5′-TCA GCT TGC TGC CTG AAG GTC AA-3′ (SEQ ID NO:4); and reverse 5′-TCA GCT TGC TGC CTG AA-3′ (SEQ ID NO:5).

Real-time PCR: Real-time PCR was performed on cDNA prepared from DNase-treated RNA. Aire, insulin2, GAD67 and cyclophilin primers were used as described (2). For VM, we used the ABI Taqman Gene Expression Assay kit (Applied Biosystems No. Mm00723516_m1). Reactions were run on a sequence detection system machine (HT7900; Applied Biosystems). For the analysis of target gene expression from thymic stromal cDNA, the standard curve method was used.

Fusion protein vectors: Recombinant proteins were produced by cloning full length cDNA sequences into the pMAL-C2X system. Lung cDNA was prepared as above. The VM primers used to generate the full length cDNA with restriction sites for subcloning were: forward 5′-CGA ATT CTC AGC TTG CTG CCT GAA GGT CAA-3′ (SEQ ID NO:6); and reverse 5′-ATG TCG ACT CAG CTT GCT GCC TGA A-3′ (SEQ ID NO:7). PCR products were subcloned into pMAL-C2X via restriction digestion, ligated and transformed into BL-21 bacteria for protein expression; protein was harvested according to the manufacturer's instructions (pMAL system, New England Biolabs).

Prediction and selection of VM peptides: An online computer program was used that relies on Average Relative Binding (ARB) matrices to predict peptide sequences that bind to MHC molecules (4). H-2 IA^(d) was selected as the matrix platform, the predicted IC₅₀ function was set as linear and the IC₅₀ (nM) cut off value was set at 5000.

ELISPOT analysis: CD4⁺ T cells from Aire^(o/o) or Aire^(+/+) mice BALB/c mice were isolated by cell sorting on a cytometer (MoFlo; Dako). The release of IFN-γ by CD4⁺ T cells was measured by ELISPOT assay. In brief, plates (Immunospot M200; BD Biosciences) were coated with 2 μg/m1 of anti-mouse IFN-γ mAb (BD Biosciences) and incubated overnight at 4° C. The plates were washed with PBS and blocked with DMEM containing 10% FCS for 2 h at 37° C. The CD4+ T cells and irradiated APCs from syngeneic mice were plated at a ratio of 1:4 and VM-MBP and MBP proteins added. The plates were incubated for 24 hrs at 37° C. in 10% CO₂ incubator. At the completion of the incubation, the plates were washed thoroughly with PBS before adding 2 μg/ml of biotin-labeled IFN-γ mAb (2 μg/ml; BD Biosciences) and incubating for 2 hrs at 4° C. After further incubation with avidin-horseradish peroxidase (1:100 dilutions; BD Biosciences) for 1 h at room temperature, the plates were developed using substrate solution (BD Biosciences). Positive spots in the plate membranes were examined using an ELISPOT reader system (Transtec; Cell Technology). The number of spot-forming cells was the average number of spots in duplicate wells.

Immunization: Six to 8 week old BALB/c Aire^(+/+) mice were immunized subcutaneously on the back in 4 spots with 100 μg of VM-MBP or MBP in an emulsion of 100 μL CFA supplemented with mycobacterium tuberculosis H37RA (4 mg/uL), using a modified EAE protocol (5). Re-immunizations using 100 μg of VM-MBP or MBP in 100 μL IFA were given 1 and 4 weeks after first immunization. Mice were sacrificed 4 weeks after the last immunization and the organs analyzed for histology.

Adoptive transfer of VM specific T cells: BALB/c Aire^(+/+) mice were immunized with 100 μg VM-111 (NLEGMLADVLNTVES set forth as SEQ ID NO:8) or the chicken ovalbumin peptide 323-339 (ISQAVHAAHAEINEAGR set forth as SEQ ID NO:9) mixed in complete Freund's adjuvant (CFA) in 4 different spots on the back. Ten days after immunization, lymph nodes and spleen were harvested. Cells were activated in vitro with 5 μg/mL of peptide in DMEM complete containing 10% FCS. After 4 days, lymphocytes were harvested by density centrifugation gradient and 20×10⁶ cells in PBS were transferred into each 6-8 week old BALB/c SCID mouse via tail vein injection.

Statistics: Data was analyzed with Prism software (GraphPad) using unpaired t-tests with a two-tailed 95% confidence interval and P≦0.05 considered significant.

Generation of ³⁵S-Radiolableled VM, LPLUNC and KCNRG- and autoantibody assay: We used full-length cDNA clones for VM (isolated as above), human LPLUNC1 (C20orf114, ATCC No. MGC-14597) and KCNRG (ATCC No. MGC-40406) for in vitro transcription and translation and labeling with ³⁵S-methionine using the TNT system kit (Promega). The ³⁵S-radiolabeled proteins immunoprecipitated with serum or positive control antibodies to LPLUNC1 (Abnova No. H00092747-B01P, Sigma No. HPA024256) were aliquoted in triplicate in 96-well PVDF filtration plates (Millipore). In each well, 20,000 counts per minute (cpm) of ³⁵S-proteins were used for immunoprecipitation. The radioactivity of the immunoprecipitated material was evaluated with the use of a liquid scintillation counter (Beckman Coulter). The autoantibody index was calculated as follows: [cpm in the unknown sample—cpm in the negative standard]÷[cpm in the positive standard−cpm in the negative standard]×100).

Human subjects: All patients and controls were included in the study only after we had obtained informed written consent. The study protocol was reviewed and approved by the institutional review board at UCSF.

Proliferation measurements by thymidine incorporation: Lymphocytes were harvested from spleen and lymph nodes of peptide-immunized mice (see above) and cultured with 5 μg/mL of either VM-111 or OVA 323-339 peptide, for 4 days in humidified 37° C., 10% CO₂ incubator. Cultures were pulsed with 1.0 μCi of [³H] thymidine at 18 hours before termination of culture, harvested using an automated multiwall harvester and counted using a scintillation counter.

Glycosidase Treatment: BAL fluid from SCID lungs lavaged with PBS was treated with N-Glycosidase F according to the manufacturer's protocol (New England Biolabs, No. P0704L).

Mass spectrometry (MS):Protein samples excised from the gel were directly submitted to the Stanford University Protein and Nucleic Acid facility for mass mapping where the samples were subjected to tryptic digestion and mass analysis of the resulting peptides. Mass mapping was performed on an Applied Biosystems 4700 Proteomics Analyzer, a MALDI mass spectrometer that provides tandem (MS/MS) time-of-flight (TOF) optics to provide peptide structural information, in addition to high-accuracy MS data (6).

Results and Discussion

Interstitial lung disease in Aire^(o/o) mice and an APS1 patient. In order to determine the pattern of lung disease in Aire^(o/o) mice BALB/c, NOD and C57BL/6 (B6) Aire^(o/o) and Aire^(+/+) mice were sacrificed at various ages. The lungs were analyzed for histology by hematoxylin and eosin (H&E) staining. At early ages, the histologic pattern of disease was identical in mice in all strains. The infiltrates were comprised of mononuclear cells in a peribronchovascular distribution. Older BALB/c and NOD mice developed progressive and often severe disease. The mononuclear infiltrates extended into the lung parenchyma and resulted in a temporally homogeneous, mild to moderate cellular interstitial pneumonia that often reached the pleural surface. A prior study reported that lung disease could be induced by the adoptive transfer of Aire^(o/o) splenocytes into immunodeficient SCID mice suggesting that the pulmonary infiltrates are autoimmune in nature (19).

APS1 patients can develop an autoimmune lung disease that is pathologically similar to the infiltrates in the mice (12). The histologic lung pathology Aire^(o/o) mice was compared to a lung biopsy specimen from a patient with APS1 and a history of pulmonary disease. The human lung biopsy demonstrated mononuclear infiltrates surrounding small and medium sized airways similar to infiltrates in Aire^(o/o) mice. The H&E stains also revealed a cellular interstitial pneumonia that mirrored findings in older NOD and BALB/c Aire^(o/o) mice. Stains for acid fast bacilli, bacteria and fungal elements were negative. These results demonstrated that Aire^(o/o) mice exhibit a bronchiolitis that in older NOD and BALB/c Aire^(o/o) animals progresses to a cellular interstitial pneumonia. This histopathology is similar to that in the biopsy from the APS1 subject with pulmonary disease.

Next the lung infiltrating and disease causing cells were identified. To this end, lungs from Aire^(o/o) mice were prepared as frozen sections and immunohistochemistry staining was performed. Within the peribronchovascular infiltrates, the cells stained primarily for CD4, although B cells (indicated by B220 staining) were also present in significant numbers. Several mice with severe disease developed organized lymphoid structures resembling bronchus-associated lymphoid tissue (BALT). This finding is consistent with reports identifying BALT in settings of chronic inflammation, including autoimmune disease (20). The majority of cells residing within the pulmonary interstitium were CD4⁺ cells, while CD8⁺ cells were present at lower numbers Immunohistochemistry of tissue sections from the APS1 patient revealed an early germinal center with a significant number of CD20⁺ B cells. CD4⁺ cells appeared slightly more abundant than the CD8⁺ cells.

Further characterization of the mouse cells by intracellular cytokine staining was performed to determine the effector mechanisms important for inducing lung disease. Lymphocytes from lungs of BALB/c and NOD Aire^(o/o) mice were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin and analyzed by flow cytometry. The cell counts confirmed that the majority of cells were CD4⁺ T cells with B cells present in significant numbers (FIG. 2). The predominant effector cytokine produced by Aire^(o/o) lung CD4⁺ T cells was interferon γ (IFN-γ), followed by roughly equivalent levels of IL-17A and IL-10, and low amounts of IL-4 secreting cells (FIG. 1A). These results show that lung infiltrating cells in Aire^(o/o) mice are primarily TH1 polarized CD4⁺ T cells, although TH17 and TH2 cells are also present.

The predominance of lymphocytes within the lung parenchyma raised the question whether Aire^(o/o) mice exhibited a lung-specific immune response. Serum from Aire^(o/o) mice with lung disease was used to stain frozen lung sections from immunodeficient SCID mice to determine whether there was autoantibody reactivity to a lung protein. Reactivity was seen on the surface of the bronchiolar epithelium (FIG. 1B); less abundant staining occurred within cells located in the alveoli. Thus, Aire^(o/o) mice develop ILD with a histopathologic pattern similar to that seen in an APS1 patient. The lung infiltrates in Aire^(o/o) mice are comprised of TH1 polarized CD4⁺ T cells, with some cells skewed toward a TH17 phenotype. Aire^(o/o) mice harbor autoantibodies that target lung proteins located in the bronchiolar epithelium and alveolar cells, indicating a lung-specific immune response.

Vomeromodulin as a major lung autoantigen in Aire^(o/o) mice Immunoblots were performed using lung lysate prepared from immunodeficient SCID mice and these bolts were subsequently probed with serum from B6, BALB/c and NOD Aire^(o/o) mice. All strains of Aire^(o/o) mice had serum autoantibodies to an 80 kD lung protein, whereas serum from Aire^(+/+) mice did not (FIG. 3A). Immunofluorescence staining using Aire^(o/o) serum (FIG. 1B) revealed that this target antigen was located on the surface of the bronchiolar epithelium. To determine whether the protein was secreted into the airway and whether it was present in BAL fluid from the lung immunoblots were performed of BAL fluid and probed with serum from Aire^(o/o) mice. The blots revealed that the 80 kD antigen was indeed present in BAL fluid (FIG. 3B).

To determine whether other antigens were targeted during the course of disease, sera were tested from NOD Aire^(o/o) mice that were serially bled over 4 to 10 weeks, and BALB/c Aire^(o/o) mice aged 5 to 15 weeks. The 80 kD band was the predominant target, with rare evidence of other immunoreactivity, even in older animals (FIG. 3C). Taken together, these results demonstrate that the 80 kD lung protein is likely a primary lung antigen in Aire^(o/o) mice in all strains.

Identification of the 80 kD lung protein targeted in Aire^(o/o) mice. The 80 kD target antigen was immunopurified from BAL fluid, concentrated and resolved on a two-dimensional gel and stained it with coomassie. Four spots were detected, three of which migrated at 80 kD, and an additional spot at 60 kD (FIG. 3D). All four spots were analyzed by mass spectrometry. Peptide mapping from two of the three spots isolated at 80 kD spanned the entire amino acid sequence and were provisionally identified as vomeromodulin (UniProt accession Q80XI7-1) with a high degree of confidence (FIG. 3E). The discrepancy between the predicted molecular weight of vomeromodulin (VM) at 62 kD and the protein on immunoblots at 80 kD appears to be due to glycosylation, which causes VM to migrate at a higher molecular weight (FIG. 4). The 60 kD spot was identified as albumin, which is abundant in BAL fluid and probably non-specifically bound to our immunoprecipitation column.

To confirm that the provisional identification of vomeromodulin corresponded to the 80 kD protein on immunoblots, a recombinant VM protein was generated and coupled to a maltose binding protein (MBP) tag. A competition assay performed using recombinant VM (VM-MBP) revealed that the 80 kD serum reactivity on immunoblots was abolished with the addition of 0.25 μg of recombinant protein. As a control, up to 16 μg of the MBP tag were added to the same serum, but these failed to abrogate the 80 kD reactivity (FIG. 3F). Further verification that VM was the target antigen came from co-staining experiments with Aire^(o/o) serum and a rat VM anti-serum. (The sequence identity between rat and mouse VM amino acid sequences is greater than 80%). Merged images of staining with the two antibodies showed they co-localized to the apical surface of the bronchiolar epithelium and to rare cells within lung alveoli (FIG. 3G). In summary, vomeromodulin was identified as the major lung autoantigen in Aire^(o/o) mice, confirming the presence of a lung autoimmune response in this model.

Aire^(o/o) mice have T cells specific for the Aire-regulated protein vomeromodulin. Vomeromodulin is enriched in the respiratory epithelium of the rat (21). To clarify the tissue distribution of VM in the mouse, RT-PCRs for full length VM cDNA were performed on cDNA libraries of several tissues. VM cDNA could only be amplified from lung cDNA (FIG. 5A). The PCR product was excised and DNA sequencing confirmed the presence of the full coding region of VM. To determine the tissue content of the VM protein, immunoblots were performed on protein lysates of different organs (FIG. 5B). The tissues were probed with a serum sample that had proven reactivity to vomeromodulin as demonstrated through competition immunoblots (FIG. 3F). In the multi-organ immunoblot, the 80 kD reactivity was detected only in lung lysate. Taken together, these data confirm that mouse VM has a restricted expression pattern that is mainly limited to the respiratory epithelium.

VM is expressed in the thymus in an Aire-dependent manner. Aire^(o/o) mice generate a VM-specific immune response and exhibit CD4⁺ T cells within lung infiltrates. The absence of thymic VM expression in Aire^(o/o) mice therefore allows the escape of VM-specific T cells that induce lung disease. A real-time PCR for VM was conducted on cDNA from purified thymic stroma of Aire^(o/o) and Aire^(+/+) mice and VM thymic expression was found to be Aire dependent, as was the expression of insulin, a known Aire-regulated antigen (FIG. 5C). As an additional control, GAD67 was tested and it was confirmed that this Aire-independent tissue specific protein was expressed equally in thymi from Aire^(o/o) and Aire^(+/+) mice (FIG. 5C) (13). To determine whether the number of IFN-γ-producing T cells with specificity for VM was increased in Aire^(o/o) mice CD4+ T cells from Aire^(o/o) and Aire^(+/+) mice were assayed for VM specificity using an ELISPOT analysis (FIG. 5D). In this assay, Aire^(o/o) mice showed a statistically significant increase in the number of IFN-γ producing, VM-specific T cells. These data demonstrate that an Aire-mediated defect in the development of tolerance in the thymus likely leads to the release of VM-specific T cells capable of inducing lung-specific tissue damage.

Breaking tolerance to VM through adjuvant immunization. To determine if VM is a major lung autoantigen that by itself can induce pulmonary disease, an immunization protocol was devised in analogy to the experimental multiple sclerosis model Experimental Autoimmune Encephalomyelitis (EAE) (23). Using complete Freund's adjuvant (CFA) wild-type BALB/c mice we immunized with VM-MBP, followed by two additional rounds of immunization using VM-MBP in incomplete Freund's adjuvant (IFA). The mice were sacrificed and their organs analyzed for histology with H&E staining. The VM-MBP immunized mice developed lung disease similar to the spontaneous disease in Aire^(o/o) mice and this disease was limited to the lung (FIG. 6A). A cohort of BALB/c wild-type mice immunized with the MBP protein tag with the same protocol did not develop lung disease (FIG. 6B). The sera from immunized mice were tested for VM antibodies and showed VM-specific immune reactivity in all of the VM immunized mice but not in the MBP-immunized controls (FIG. 6C). These results demonstrate that inducing a break in tolerance with adjuvant immunization can activate VM-specific cells to cause a lung-restricted disease similar to the spontaneous pulmonary infiltrates observed in Aire^(o/o) mice.

Lung specific disease from the adoptive transfer of activated VM-specific cells. One hallmark of autoimmune disease is that the adoptive transfer of activated antigen-specific cells to another host transfers the disease. Therefore a protocol was devised for the adoptive transfer of VM-specific immune cells harvested from immunized wild-type mice. To facilitate in vitro activation of the cells, VM peptides were generated that by computer modeling were predicted to bind to BALB/c MHC Class II, I-Ad (24). The peptides were then screened by ELISPOT analysis to determine whether Aire^(o/o) mice had T cells specific for them. As shown in FIG. 7, Aire^(o/o) mice had IFN-γ-producing T cells specific for the 15 amino acid peptide NLEGMLADVLNTVES (VM-111 set forth as SEQ ID NO: 8). The VM-111 peptide was used to immunize wild-type BALB/c mice and in vitro activate spleen and lymph node cells from these mice in conditions favoring the growth of CD4⁺ T cells. A peptide derived from chicken ovalbumin (Ova) that is also known to bind I-A^(d) was used as our negative control for the disease transfer (FIG. 8A). A portion of cells from immunized mice was analyzed by [H³] thymidine incorporation to confirm that cells proliferated in response to our antigen (FIG. 8B). About 20×10⁶ activated cells were adoptively transferred into each of our immunodeficient SCID mice, which were sacrificed 4 to 6 weeks later. The resulting lung disease was less severe than the spontaneous disease of Aire^(o/o) mice, but the infiltrates were limited to the lung (FIGS. 8C-D) and had a similar appearance. These results show that the adoptive transfer of activated VM-specific cells can transfer lung disease and that the likely pathogenic effectors are CD4⁺ T cells.

Autoreactivity to LPLUNC1, a VM-like protein, in a patient with APS1. Based on this data supporting an autoreactive lung response in Aire^(o/o) mice, a similar response was investigated in an APS1 patient with lung disease. First, whether the patient exhibited an autoimmune response to lung tissue was tested by staining frozen sections of normal human lung with the patient's serum (FIG. 9A). The pattern of immunoreactivity was nearly identical to the pattern of staining seen using sera from Aire^(o/o) mice (FIG. 1B, and FIG. 9A). The patient's serum did not contain autoantibodies to KCNRG, a bronchial epithelial antigen recently identified in APS1 (12) (FIG. 10).

A direct human homolog of mouse VM does not exist. On the basis of surveys of the University of California Santa Cruz and Ensembl genome databases, the human gene transcript orthologous to VM is C20orf115 and is likely an unprocessed pseudogene. The organization of the human genomic region containing C20orf115 is similar to that of the mouse, and it contains the PLUNC family of proteins, including LPLUNC1 (C20orf114), the adjacent transcript upstream from the human VM pseudogene (FIG. 9C). An analysis of the domain structure of VM using the NCBI Conserved Domain Database revealed that the VM protein shares the BPI (Bactericidal/Permeability Increasing protein) superfamily domain with the PLUNC (Palate, Lung, and Nasal epithelium Carcinoma associated protein) family of proteins located in the same region on mouse chromosome 2 (FIGS. 9D-E) (25). The PLUNC proteins have not previously been implicated in autoimmunity, but, because of their similar domain structure and expression pattern to VM, the APS1 patient serum was tested against LPLUNC1, the family member with the highest levels of gene expression in the lung, in particular the bronchiolar epithelium (26, 27). An autoantibody assay revealed that the APS1 patient with lung disease had immunoreactivity to LPLUNC1 that was not seen in the healthy controls or other samples from APS1 patients without lung disease (FIG. 9F) Immunofluorescent staining of normal human lung tissue with a human LPLUNC1 antibody showed that the protein is located on the bronchiolar epithelium in a pattern similar to the LPLUNC1 distribution in the APS1 patient (FIG. 9G-H). This data shows that the lung disease in an APS1 patient closely recapitulates the lung disease in Aire^(o/o) mice, suggesting that LPLUNC1 is an important human lung autoantigen.

As shown in FIG. 11, higher titers of autoantibodies to human LPLUNC1 (e.g., elevated LPLUNC1 autoantibody index) were detected in serum of ILD patients than in normal control subjects. In this assay, the positive controls included an APS1 patient with known LPLUNC1 autoantibody reactivity, and two commercial antibodies to human LPLUNC1. The ILD sera were obtained from a randomly selected subset of the patients seen and enrolled in the clinical database of the University of California, San Francisco (UCSF) ILD clinic. The normal controls are healthy subjects who do not have known autoimmunity or ILD.

TABLE 1 UCSF Interstitial Lung Disease (ILD) Patients by Diagnosis Percentage Diagnosis (n = 548) Idiopathic Pulmonary Fibrosis 17% Sarcoiditis 12% Hypersensitivity Pneumonitis 12% Scleroderma 6% Not ILD 5% Connective Tissue Disease - Undifferentiated 5% Non-specific Interstitial Pneumonia 4% Rheumatoid Arthritis 4% Myositis 2% Other Diagnoses 14% Unclassifiable 9% Not Reported 7% Pending Diagnosis 4%

TABLE 2 Baseline Characteristics of ILD Patients 1-5 of FIG. 11 LPLUNC1 Evidence of Patient Age Sex Ab Index Diagnosis Autoimmunity 1 77 F 1.05 Non-specific Rheumatoid Interstitial Arthritis Pneumonia (CCP⁺, RA⁺) (NSIP) 2 72 M 0.33 Idiopathic No Pulmonary Fibrosis 3 46 F 0.24 NSIP and Dermato- Organizing myositis Pneumonia 4 62 F 0.23 Idiopathic No NSIP and Bronchiolitis 5 67 M 0.23 Idiopathic No Pulmonary Fibrosis Negative — — 0.03 ± 0.03 N/A No Controls

TABLE 3 Baseline Characteristics of ILD Patients of FIG. 13 Age LPLUNC Sex (years) Race Diagnosis AutoAb Index Female 76 White CTD-ILD 156 Male 44 Asian CTD-ILD 42 Male 57 White CTD-ILD 31 Male 36 White Unclassifiable 29 Male 45 White Idiopathic NSIP 28 Male 71 White Idiopathic NSIP 28 Male 77 White Unclassifiable 28 Male 67 White CTD-ILD 27 Male 68 White IPF 22 Female 44 Asian Idiopathic NSIP 22 Male 63 White CTD-ILD 20 Female 70 White Idiopathic bronchiolitis 20 Male 81 White IPF 20 Male 59 White CTD-ILD 19 Female 49 Asian CTD-ILD 19 Female 58 White COP 19 Female 82 White COP 19 Female 70 White Unclassifiable 17 Female 34 White CTD-ILD 15 Male 63 White IPF 15 Female 68 White Unclassifiable 15 Male 74 White IPF 14 Male 77 White Idiopathic bronchiolitis 12 Female 46 White CTD-ILD 11 * IPF, idiopathic pulmonary fibrosis; CTD-ILD, connective tissue disease associated-ILD; COP, cryptogenic organizing pneumonia; and NSIP, non-specific interstitial pneumonia.

FIG. 13 and Table 3 provide updates to FIG. 11 and Table 2, respectively.

Bovine lung tissue was stained using sera from an ILD patient with demonstrated positivity in the LPLUNC1 autoantibody assay. The staining showed that the patient serum reacts to the bronchiolar epithelium in a pattern similar to the LPLUNC1 distribution in the APS1 patient and in sections stained with a commercial LPLUNC1 antibody. For comparison, a control patient's sera was used to stain a serial section of tissue.

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We claim:
 1. A method for assessing whether a mammalian patient has or is predisposed to an interstitial lung disease (ILD), said method comprising: a) performing an antibody-based assay to quantitate lung autoantigen-reactive autoantibodies in a biological sample from the patient, wherein the lung autoantigen is a long palate, lung, and nasal epithelium carcinoma-associated protein 1 (LPLUNC1) or a LPLUNC1-like protein comprising the amino acid sequence at least 70% identical to SEQ ID NO:2; and b) detecting an elevated level of the lung autoantigen-reactive autoantibodies in said biological sample as compared to a control biological sample thereby determining the patient has ILD or a predisposition to ILD.
 2. The method of claim 1, wherein the lung autoantigen is a long palate, lung, and nasal epithelium carcinoma-associated protein 1 (LPLUNC 1).
 3. The method of claim 1, wherein the lung autoantigen is a LPLUNC1-like protein comprising the amino acid sequence at least 70% identical to SEQ ID NO:2.
 4. The method of claim 1, wherein the biological sample is selected from a group consisting of blood, plasma, serum, bronchial alveolar lavage (BAL) fluid, and lung tissue.
 5. The method of claim 1, wherein the patient has or is suspected of having a systemic autoimmune disease.
 6. The method of claim 5, wherein the systemic autoimmune disease is selected from the group consisting of rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosis, sarcoidosis, Wegener's granulomatosis, and autoimmune polyendocrine syndrome type 1 (APS-1).
 7. The method of claim 1, wherein the antibody-based assay is selected from the group consisting of ELISA, Western blotting, immunofluorescence analysis, flow cytometry, and antibody microarray.
 8. The method of claim 7, wherein said antibody-based assay comprises a first reagent that specifically binds to said lung autoantigen-reactive autoantibodies, and a second reagent for detecting said autoantibodies, wherein said first reagent is the lung autoantigen or a cell expressing the lung autoantigen, and said second reagent comprises a secondary antibody that is reactive with constant regions of the autoantibodies.
 9. The method of claim 1, further comprising performing one or both of a pulmonary function test and a high resolution computed tomography scan on said patient.
 10. The method of claim 1, further comprising: c) administering a treatment to the patient when the elevated level of the lung autoantigen-reactive autoantibodies is detected.
 11. The method of claim 10, wherein said treatment is selected from the group consisting of a corticosteroid, cyclophosphamide, mycophenolate mofetil, and azathioprine.
 12. The method of claim 10, wherein said treatment comprises a mucosal tolerance regimen comprising dispensing a formulation to the patient by an oral or an intra-nasal route, wherein the formulation comprises a pharmaceutically acceptable excipient, and an effective amount of the lung autoantigen, or a peptide derived therefrom.
 13. The method of claim 10, wherein said treatment comprises a parenteral tolerance regimen comprising dispensing a formulation to the patient by intravenous or subcutaneous injection, wherein the formulation comprises an effective amount of the lung autoantigen, a peptide derived therefrom, or a nucleic acid encoding the lung autoantigen in operable combination with a regulatory sequence.
 14. The method of claim 10, wherein said treatment comprises an antigen-coupled cell tolerance regimen comprising dispensing a formulation to the patient by intravenous injection, wherein the formulation comprises an effective amount of the lung autoantigen, or a peptide derived therefrom, wherein the autoantigen or the peptide is coupled to ethylene carbodiimide-fixed, autologous antigen presenting cells.
 15. The method of claim 10, wherein said treatment comprises a regulatory T cell regimen comprising dispensing a formulation to the patient by intravenous injection, wherein the formulation comprises an effective amount of ex vivo-expanded lung autoantigen-specific regulatory T cells. 